Currently, there exist at least three technical barriers to growing confluent neointima on synthetic vascular grafts through BC seeding: BC must be (1) seeded efficiently onto the lumen of vascular grafts, leading to a confluent monolayer; (2) adherent to the graft surface. to avoid being stripped from the surface upon the onset of blood flow; and (3) quiescent with an anti-thrombotic profile, while not releasing factors that lead to thrombosis, intimal hyperplasia, and leukocyte adhesion. Overall goal of this competitive renewal is to promote the rapid adaptation of seeded BC to their in vivo quiescent lumenal status of confluence and release of anti-thrombotic mediators. We hypothesize that this can be achieved through a combination of firm adhesion and flow preconditioning. The project is divided into "applied" and "mechanistic" research efforts, where the mechanistic studies are intended to complement and guide the future development of graft surfaces. The "applied" objective is to determine the conditions of lumenal shear forces (using flow preconditioning) and ablumenal adhesive interactions (using integrin-mediated and integrin- independent ligands) that signal seeded BC to adopt a confluent and anti-thrombotic condition. This involves in vitro assessment of BC adhesion, spreading, alignment, focal contact formation, anti-thrombotic mediator release, and leukocyte/platelet adhesion (Specific Aim l), and in vivo assessment of implanted BC-seeded vascular grafts for patency, number of adherent BC, thrombus formation, intimal thickness, platelet and leukocyte adhesion (Specific 2). The relationship between lumenal and ablumenal signaling experienced by seeded BC will be deduced using two in vitro "mechanistic" approaches that elucidate the relationship between apical physical forces and basal BC adhesion upon BC function (Specific Aims 3 and 4). The mechanistic studies will "get under the hood" of the seeded BC by specifically and precisely modulating apical and basal signaling using a novel combination of atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and ligand microstamping.